Periodic Reporting for period 4 - BONEPHAGY (Defining the role of the FGF – autophagy axis in bone physiology)
Periodo di rendicontazione: 2021-07-01 al 2022-12-31
The fibroblast growth factor (FGF) signaling pathways have been recognized as essential regulators of vertebrate skeletal development. The FGF family comprises 18 secreted proteins that bind to and activate 4 receptor tyrosine kinase molecules (FGFRs), inducing receptor dimerization and activation, and induction of multiple intracellular effectors. Mutations in FGF ligands and receptors result in at least 14 different types of human genetic disorders characterized by defective skeletal development and growth. A notable example is achondroplasia (ACH), the most common form of dwarfism (frequency about 1:15000), which is prevalently due to a G380R aa substitution in FGFR3, a negative regulator of bone growth, resulting in a gain-of-function. Consistently, loss-of-function mutations in FGFR3 cause skeletal overgrowth, camptodactyly, tall stature, and hearing loss (CATSHL) syndrome. Genome-wide association (GWA) studies have also identified variants in FGFR4 gene that are associated to human growth. In addition, FGF9 and FGF18 ligands regulate bone and cartilage homeostasis, thus playing a role during fracture bone repair and osteoarthritis. Many pathways are controlled by FGF signaling in chondrocytes and osteoblasts, and their deregulation most likely contribute to the pathogenesis of FGF-related skeletal dysplasia. Thus, the identification of new intracellular effectors of FGF signaling in cartilage and bone is an important ongoing topic of research, not only to discover new mechanisms regulating bone growth and homeostasis, but also to provide new potential therapeutic avenues for the treatment of FGF-skeletal dwarfism. The work proposed in the grant BONEPHAGY aims to:
a) Define the molecular mechanisms by which FGF signaling control autophagy FGF-autophagy axis.
b) To determine the physiological relevance of autophagy regulation by FGF signaling.
c) Develop new therapeutic approaches for disorders due to mutations in the FGF signaling pathway.
a) Define the molecular mechanisms by which FGF signaling control autophagy FGF-autophagy axis.
b) To determine the physiological relevance of autophagy regulation by FGF signaling.
c) Develop new therapeutic approaches for disorders due to mutations in the FGF signaling pathway.
My laboratory demonstrated that FGF signaling is a major regulator of autophagy during post-natal bone growth (26595272). Autophagy is a catabolic process involved in the degradation and turnover of intracellular substrates. It relies on the biogenesis of autophagosomes, double membrane vesicles that sequester cytosolic substrates and target them to the lysosomes for degradation by hydrolases. The work proposed in BONEPHAGY aims to: i) define the molecular mechanisms by which FGF signaling controls autophagy, ii) determine the physiological relevance of autophagy regulation by FGF signaling, iii) develop new therapeutic approaches for disorders due to mutations in FGF signaling pathway.
We identified the FGF signaling as a major regulator of autophagy during post-natal bone growth. Mice lacking autophagy showed endoplasmic reticulum (ER) enlargement and misfolded collagen accumulation into the ER, thus leading to disorganized fibrillary network in the extracellular matrix (ECM) and impaired bone growth (26595272). Notably, animal models of Lysosomal Storage Disorders (LSDs), which showed autophagy flux impairment, also showed delayed collagen trafficking through the ER and skeletal defects (28872463). Thus, my laboratory has discovered that collagen is a new autophagy substrate (26595272; 28872463; 30559329). We have also characterized the molecular mechanism underling autophagy activation by FGF signaling in chondrocytes. We have found that FGF18 activates FGFR3 and FGFR4 receptors leading to phosphorylation and degradation of Insulin Receptor Substrate 1 (IRS1), a key player of the insulin signaling pathway, that in turn induced mTORC1 inactivation and the nuclear translocation of the transcription factors TFEB and TFE3, master regulators of autophagy and lysosomal genes. Interestingly, TFEB/3 induced lysosomes biogenesis, but also enhanced the expression of FAM134B gene, which encodes a selective receptor for the autophagy of the ER (or ER-phagy), a specific form of autophagy in which the receptor brings a portion of the ER membrane to autophagosomes thanks to the LC3-Interaction Region (LIR) domain. In chondrocytes the FGF-TFEB-FAM134B axis transcriptionally controls the process of ER-phagy contributing to the quality control of the ER. Animal models lacking ER-phagy (Fam134b-/- Medaka fishes and FGFR3-/-;FGFR4-/- mice) showed ER accumulation of collagen, ER enlargement and skeletal defects (32716134).
My lab has further identified a new function of FAM134B-mediated ER-phagy as a quality control system for misfolded cargoes at the ER, by characterizing how autophagy selectively recognizes misfolded procollagens in the ER lumen. Using different approaches (siRNA, CRISPR-Cas9 or KO-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells) we have found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with FAM134B, which in turn binds the autophagosome membrane-associated protein LC3 through the LIR domain and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation (30559329). Thus, we have defined a crosstalk between the ER quality control machinery and the autophagy pathway in selectively disposing of proteasome-resistant misfolded clients from the ER.
In addition, my lab has identified a post-translational mechanism unique for another FAM134 family member, FAM134C. In fed conditions, FAM134C is phosphorylated by casein kinase 2 (CK2) which negatively affects binding affinity to the autophagy proteins LC3 (36044577). During starvation, mTORC1 inhibition limits FAM134C phosphorylation by CK2, hence promoting receptor activation and ER-phagy. We generated a novel tool to study ER-phagy in vivo (FAM134C KO mice), used to demonstrate the physiological relevance of FAM134C phosphorylation during starvation-induced ER-phagy in liver lipid metabolism (36044577). All in all, in those years, my laboratory has discovered that the ER-phagy process is fundamental for the ER quality control in vitro and in vivo, and defined the biological pathways underlining transcriptional and post-translational regulation of ER-phagy.
Notably, as a proof of concept, we have already demonstrated that the pharmacological enhancement of autophagy ameliorates the skeletal manifestation in 2 different mouse models of lysosomal storage disorders (28872463). Furthermore, we are currently investigating whether the enhancement of autophagy, ER-phagy and lysosomal catabolism is beneficial for the treatment of genetic skeletal disorders.
We identified the FGF signaling as a major regulator of autophagy during post-natal bone growth. Mice lacking autophagy showed endoplasmic reticulum (ER) enlargement and misfolded collagen accumulation into the ER, thus leading to disorganized fibrillary network in the extracellular matrix (ECM) and impaired bone growth (26595272). Notably, animal models of Lysosomal Storage Disorders (LSDs), which showed autophagy flux impairment, also showed delayed collagen trafficking through the ER and skeletal defects (28872463). Thus, my laboratory has discovered that collagen is a new autophagy substrate (26595272; 28872463; 30559329). We have also characterized the molecular mechanism underling autophagy activation by FGF signaling in chondrocytes. We have found that FGF18 activates FGFR3 and FGFR4 receptors leading to phosphorylation and degradation of Insulin Receptor Substrate 1 (IRS1), a key player of the insulin signaling pathway, that in turn induced mTORC1 inactivation and the nuclear translocation of the transcription factors TFEB and TFE3, master regulators of autophagy and lysosomal genes. Interestingly, TFEB/3 induced lysosomes biogenesis, but also enhanced the expression of FAM134B gene, which encodes a selective receptor for the autophagy of the ER (or ER-phagy), a specific form of autophagy in which the receptor brings a portion of the ER membrane to autophagosomes thanks to the LC3-Interaction Region (LIR) domain. In chondrocytes the FGF-TFEB-FAM134B axis transcriptionally controls the process of ER-phagy contributing to the quality control of the ER. Animal models lacking ER-phagy (Fam134b-/- Medaka fishes and FGFR3-/-;FGFR4-/- mice) showed ER accumulation of collagen, ER enlargement and skeletal defects (32716134).
My lab has further identified a new function of FAM134B-mediated ER-phagy as a quality control system for misfolded cargoes at the ER, by characterizing how autophagy selectively recognizes misfolded procollagens in the ER lumen. Using different approaches (siRNA, CRISPR-Cas9 or KO-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells) we have found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with FAM134B, which in turn binds the autophagosome membrane-associated protein LC3 through the LIR domain and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation (30559329). Thus, we have defined a crosstalk between the ER quality control machinery and the autophagy pathway in selectively disposing of proteasome-resistant misfolded clients from the ER.
In addition, my lab has identified a post-translational mechanism unique for another FAM134 family member, FAM134C. In fed conditions, FAM134C is phosphorylated by casein kinase 2 (CK2) which negatively affects binding affinity to the autophagy proteins LC3 (36044577). During starvation, mTORC1 inhibition limits FAM134C phosphorylation by CK2, hence promoting receptor activation and ER-phagy. We generated a novel tool to study ER-phagy in vivo (FAM134C KO mice), used to demonstrate the physiological relevance of FAM134C phosphorylation during starvation-induced ER-phagy in liver lipid metabolism (36044577). All in all, in those years, my laboratory has discovered that the ER-phagy process is fundamental for the ER quality control in vitro and in vivo, and defined the biological pathways underlining transcriptional and post-translational regulation of ER-phagy.
Notably, as a proof of concept, we have already demonstrated that the pharmacological enhancement of autophagy ameliorates the skeletal manifestation in 2 different mouse models of lysosomal storage disorders (28872463). Furthermore, we are currently investigating whether the enhancement of autophagy, ER-phagy and lysosomal catabolism is beneficial for the treatment of genetic skeletal disorders.
We have characterized a novel signaling pathway that is activated by FGFs in chondrocytes. These data might be important for the identification of potential therapeutic targets for treating genetic skeletal dysplasia and growth disorders.
We have identified the molecular mechanisms mediating procollagen clearance from the ER via autophagy. We have provided proof of principle that modulation of autophagy might be beneficial for the treatment of skeletal abnormalities in lysosomal storage disorders. In the next years we plan to investigate whether manipulation of the signaling pathway we have identified in chondrocytes might be beneficial for the treatment of FGF-related skeletal disorders.
We have identified the molecular mechanisms mediating procollagen clearance from the ER via autophagy. We have provided proof of principle that modulation of autophagy might be beneficial for the treatment of skeletal abnormalities in lysosomal storage disorders. In the next years we plan to investigate whether manipulation of the signaling pathway we have identified in chondrocytes might be beneficial for the treatment of FGF-related skeletal disorders.